Aeroplane wing, a new recessive autosomal phenotypic marker in the malaria vector, Anopheles stephensi Liston.

A novel and distinct mutant with a phenotype, aeroplane wing (ae) is reported for the first time in the urban malaria vector Anopheles stephensi. The main aim of this study was to establish the mode of inheritance of the ae gene performing genetic crossings between the mutants and wild types. These mutants show extended open wings that are visible to naked eyes in both the sexes. Mutants were first noticed in a nutritionally stressed isofemale colony. Strategic genetic crosses revealed that the ae gene is a recessive, autosomal, and monogenic trait having full penetrance with uniform expression in its adult stage. Egg morphometric analysis confirmed that these mutants were intermediate variant. No significant differences were observed in the wing venation and size of ae mutants compared to their control parental lines. Further cytogenetic analysis on the ovarian polytene chromosome of ae mutant showed an inversion (3Li) on the 3L arm like its parental line. This ae mutant would be a prominent marker and could be useful to study the functions of related specific genes within its genome.

Enrichment of phenotype among biological forms of Anopheles stephensi Liston through establishment of isofemale lines.

BACKGROUND: Vector management programs rely on knowledge of the biology and genetic make-up of mosquitoes. Anopheles stephensi is a major invasive urban malaria vector, distributed throughout the Indian subcontinent and Middle East, and has recently been expanding its range in Africa. With the existence of three biological forms, distinctly identifiable based on the number of ridges on eggs and varying vectorial competence, An. stephensi is a perfect species for developing isofemale lines, which can be tested for insecticide susceptibility and vectorial competence of various biological forms. METHODS: We describe key steps involved in establishment and validation of isofemale lines. Isofemale colonies were further used for the characterization of insecticide susceptibility and differential vector competence. The results were statistically evaluated through descriptive and inferential statistics using Vassar Stat and Prism GraphPad software packages. RESULTS: Through a meticulous selection process, we overcame an initial inbreeding depression and found no significant morphometric differences in wings and egg size between the parental and respective isofemale lines in later generations. IndCh and IndInt strains showed variations in resistance to different insecticides belonging to all four major classes. We observed a significant change in vectorial competence between the respective isofemale and parental lines. CONCLUSIONS: Isofemale lines can be a valuable resource for characterizing and enhancing several genotypic and phenotypic traits. This is the first detailed report of the establishment of two isofemale lines of type and intermediate biological forms in Anopheles stephensi. The work encompasses characterization of fitness traits among two lines through a transgenerational study. Furthermore, isofemale colonies were established and used to characterize insecticide susceptibility and vector competence. The study provides valuable insights into differential susceptibility status of the parental and isofemale lines to different insecticides belonging to the same class. Corroborating an earlier hypothesis, we demonstrate the high vector competence of the type form relative to the intermediate form using homozygous lines. Using these lines, it is now possible to study host-parasite interactions and identify factors that might be responsible for altered susceptibility and increased vector competence in An. stephensi biological forms that would also pave the way for developing better vector management strategies.

Identification of a TNF-TNFR-like system in malaria vectors (Anopheles stephensi) likely to influence Plasmodium resistance.

Identification of Plasmodium-resistance genes in malaria vectors remains an elusive goal despite the recent availability of high-quality genomes of several mosquito vectors. Anopheles stephensi, with its three distinctly-identifiable forms at the egg stage, correlating with varying vector competence, offers an ideal species to discover functional mosquito genes implicated in Plasmodium resistance. Recently, the genomes of several strains of An. stephensi of the type-form, known to display high vectorial capacity, were reported. Here, we report a chromosomal-level assembly of an intermediate-form of An. stephensi strain (IndInt), shown to have reduced vectorial capacity relative to a strain of type-form (IndCh). The contig level assembly with a L50 of 4 was scaffolded into chromosomes by using the genome of IndCh as the reference. The final assembly shows a heterozygous paracentric inversion, 3Li, involving 8 Mbp, which is syntenic to the extensively-studied 2La inversion implicated in Plasmodium resistance in An. gambiae involving 21 Mbp. Deep annotation of genes within the 3Li region in the IndInt assembly using the state-of-the-art protein-fold prediction and other annotation tools reveals the presence of a tumor necrosis factor-alpha (TNF-alpha) like gene, which is the homolog of the Eiger gene in Drosophila. Subsequent chromosome-wide searches revealed homologs of Wengen (Wgn) and Grindelwald (Grnd) genes, which are known to be the receptors for Eiger in Drosophila. We have identified all the genes in IndInt required for Eiger-mediated signaling by analogy to the TNF-alpha system, suggesting the presence of a functionally-active Eiger signaling pathway in IndInt. Comparative genomics of the three type-forms with that of IndInt, reveals structurally disruptive mutations in Eiger gene in all three strains of the type-form, suggesting compromised innate immunity in the type-form as the likely cause of high vectorial capacity in these strains. This is the first report of the presence of a homolog of Eiger in malaria vectors, known to be involved in cell death in Drosophila, within an inversion region in IndInt syntenic to an inversion associated with Plasmodium resistance in An. gambiae.

The genome trilogy of Anopheles stephensi, an urban malaria vector, reveals structure of a locus associated with adaptation to environmental heterogeneity.

Anopheles stephensi is the most menacing malaria vector to watch for in newly urbanising parts of the world. Its fitness is reported to be a direct consequence of the vector adapting to laying eggs in over-head water tanks with street-side water puddles polluted by oil and sewage. Large frequent inversions in the genome of malaria vectors are implicated in adaptation. We report the genome assembly of a strain of An. stephensi of the type-form, collected from a construction site from Chennai (IndCh) in 2016. The genome reported here with a L50 of 4, completes the trilogy of high-resolution genomes of strains with respect to a 16.5 Mbp 2Rb genotype in An. stephensi known to be associated with adaptation to environmental heterogeneity. Unlike the reported genomes of two other strains, STE2 (2R+b/2Rb) and UCI (2Rb/2Rb), IndCh is found to be homozygous for the standard form (2R+b/2R+b). Comparative genome analysis revealed base-level details of the breakpoints and allowed extraction of 22,650 segregating SNPs for typing this inversion in populations. Whole genome sequencing of 82 individual mosquitoes from diverse geographical locations reveal that one third of both wild and laboratory populations maintain the heterozygous genotype of 2Rb. The large number of SNPs can be tailored to 1740 exonic SNPs enabling genotyping directly from transcriptome sequencing. The genome trilogy approach accelerated the study of fine structure and typing of an important inversion in An. stephensi, putting the genome resources for this understudied species on par with the extensively studied malaria vector, Anopheles gambiae. We argue that the IndCh genome is relevant for field translation work compared to those reported earlier by showing that individuals from diverse geographical locations cluster with IndCh, pointing to significant convergence resulting from travel and commerce between cities, perhaps, contributing to the survival of the fittest strain.

Cumulative human exposure and environmental occurrence of phthalate esters: A global perspective.

In the current investigation, the distribution and extent of human exposure of phthalate esters (PAEs) have been reported on global extent based on computed indices. The proposed indices were calculated based on environmental concentrations, toxicity, occurrence, environmental fate, and transport of PAEs. The cumulative phthalate exposure index (PEI) based on the phthalate pollution index (PPI) was mapped on a global scale based on the existing data reported in the literature. The results revealed that the PAEs are heterogeneously distributed globally, and about 30% of total environmental PAEs are ultimately exposed to the average human being. The comparative distribution of PAEs in various environmental compartments including urban-rural, indoor-outdoor, home-dormitory-classroom, and their exposure likelihood were studied based on indices. More than 90% of total human exposure of PAEs was observed to be from indoor environmental compartments. Significantly high exposure was observed in the urban population as compared to the rural population. About 70% of the total phthalate pollution sub-index of dust was observed from home followed by a classroom of ∼15% and then a dormitory of ∼10%. In addition, the indices were equated with the current human development index (HDI), gross national income (GNI), and exposure of particulate matter of each country. Based on current findings, the population living in the areas where >20 µg/m3 of particulate matter has been reported are possibly exposed with higher PAEs. The indices were highly diversified at high HDI (0.9) values and between 40,000 and 50,000 $ per capita income due to different usage of phthalate-containing products, disposal, and extensive monitoring work carried out by the developed and developing countries.

Increasing the potential for malaria elimination by targeting zoophilic vectors.

Countries in the Asia Pacific region aim to eliminate malaria by 2030. A cornerstone of malaria elimination is the effective management of Anopheles mosquito vectors. Current control tools such as insecticide treated nets or indoor residual sprays target mosquitoes in human dwellings. We find in a high transmission region in India, malaria vector populations show a high propensity to feed on livestock (cattle) and rest in outdoor structures such as cattle shelters. We also find evidence for a shift in vector species complex towards increased zoophilic behavior in recent years. Using a malaria transmission model we demonstrate that in such regions dominated by zoophilic vectors, existing vector control tactics will be insufficient to achieve elimination, even if maximized. However, by increasing mortality in the zoophilic cycle, the elimination threshold can be reached. Current national vector control policy in India restricts use of residual insecticide sprays to domestic dwellings. Our study suggests substantial benefits of extending the approach to treatment of cattle sheds, or deploying other tactics that target zoophilic behavior. Optimizing use of existing tools will be essential to achieving the ambitious 2030 elimination target.

Development and evaluation of a single-step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species.

OBJECTIVE: To develop a single-step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic/epidemic areas and to detect the most abundant species by the multiplex PCR. METHODS: Aquatic stages of different mosquito species were sampled by inspecting artificial and natural breeding sites in domestic and peridomestic areas. DNA was isolated from different stages of the three Aedes species. Using novel primers based on 18S rDNA sequence, a single-step multiplex PCR was developed to clearly distinguish the three Aedes species. It was then evaluated in the aquatic stages of Aedes species collected from different areas. RESULTS: A total of 1150 aquatic stages were collected from 294 breeding spots, of which 156 contained Aedes species. Discarded tires were the major breeding spots of Aedes species. The aquatic stages were clustered into 230 pools; Ae. albopictus was detected in the largest number of pools, followed by Ae. aegypti and Ae. vittatus. CONCLUSIONS: The Multiplex PCR clearly differentiated the aquatic stages of the three Aedes species and detected that Ae. albopictus was most profuse in different breeding spots surveyed, hence indicating to be the main vector in this region. So control measures can be designed against Ae. albopictus at an early stage to prevent any arboviral outbreak. This method is a convenient tool for precise identification of Aedes vectors during entomological surveys in arbovirus endemic/epidemic areas where several species coexist.

Molecular identification and phylogeny of Myzomyia and Neocellia series of Anopheles subgenus Cellia (Diptera: Culicidae).

Any biological study is only meaningful if the concerned organism is accurately identified; this is particularly important in vector-borne disease studies where correct and precise identification of the target species has medical and practical implications, such as in vector control. The Myzomyia series is divided into four groups including the Funestus group, which consists of five subgroups, i.e. Aconitus, Culicifacies, Funestus, Minimus, Rivulorum, and the Neocellia series, which is divided into three groups Annularis, Jamesii and Maculatus. Members of the Funestus group of Myzomyia and the Annularis group of the Neocellia series are difficult to identify because of the morphological overlap that exists within the groups. Therefore a multiplex polymerase chain reaction (PCR) assay was developed based on the sequence of the D3 region of 28S rDNA to distinguish between four members (An. fluviatilis, An. culicifacies, An. varuna and An. aconitus) of three subgroups (Minimus, Aconitus, Culicifacies) of the Funestus group of Myzomyia and three members (An. annularis, An. pallidus and An. philippinensis) of the Annularis group of the Neocellia series of the Anopheles subgenus Cellia, prevalent in Orissa, India. Polymorphism present on the D3 region of rDNA allowed the development of a species-specific primer that when combined with two universal primers lead to a simple and sensitive multiplex allele-specific polymerase chain reaction (AS-PCR) assay. This assay can be applied as an unbiased confirmatory method for the identification of morphological variants, imperfectly preserved specimens and life stages for which taxonomic keys do not allow a definitive species determination. Finally, phylogenetic relationships between the members of the two series were determined using D3 sequence data. The phylogenetic relationships inferred from maximum parsimony and the neighbour joining analysis separated two distinct monophyletic clades, one consisting of species of Myzomyia and other of species of the Neocellia series. The molecular phylogeny obtained in this work matches with that of the classical morphological taxonomy reasonably well, with proper species arrangements.

Analysis of the phylogenetic relationship of Anopheles species, subgenus Cellia (Diptera: Culicidae) and using it to define the relationship of morphologically similar species.

Studies on the relationship of various vectors and non-vectors of malaria from the evolutionary point of view are important. Use of molecular methods to define phylogeny helps to understand the interrelationship among the members of the anophelines and elucidate the ambiguity that has arisen from improper classification. It could also help to design molecular markers for species differentiation, particularly in those which pose difficulty when classified, based on morphological features. In the present study, the phylogenetic relationships among the species of the anophelines of subgenus Cellia are inferred from the mitochondrial genes COI and COII, the ribosomal RNA gene, in particular the D3 region, and Internal Transcribed Spacer 2 (ITS2) region. The molecular phylogeny obtained in this work matches with that of the classical morphological taxonomy reasonably well, and was useful in properly defining species positions and resolving the ambiguity that normally arises due to morphological taxonomy. The correct arrangement of the various anopheline taxa as per the traditional morphological character-based classification of anophelines was there when we considered the D3 region of 28S rRNA gene and ITS2 region of rDNA. However, the arrangement of the taxa did not match with that of the morphological classification in some aspects, when we considered the COI and COII region of mitochondrial DNA. It may have been due to the variable degree of the rate of evolution of the different genes within the organism. Thus, a proper selection of those particular genes that evolve at the rate that is reflected at the species differentiation level, could help to construct the correct phylogenetic relationship among the anophelines and could be used to correlate with the grouping pattern done from the morphological perspective.

A unique methodology for detecting the spread of chloroquine-resistant strains of Plasmodium falciparum, in previously unreported areas, by analyzing anophelines of malaria endemic zones of Orissa, India.

Generally, clinical data is referred to study drug-resistance patterns of Plasmodium falciparum in an area. This is only possible after a clear manifestation of drug-resistance parasites inside the human host, and thereafter detection by healthcare persons. The detection of spread of drug-resistant P. falciparum in a population, before any pathological symptoms detected in humans is possible by analyzing the anopheline vectors, transmitting malaria. In the present study we implemented a new strategy to detect the spread of chloroquine-resistant (CQR) strains of P. falciparum by the major malaria vectors prevalent in selected endemic regions of Orissa, India. We screened P. falciparum positive vectors by using polymerase chain reaction (PCR)-based assay and thereafter detected K76T mutation in the Pfcrt gene, the chloroquine-resistance marker, of parasites present within the vectors. This study showed higher transmission rate of chloroquine-resistant P. falciparum parasites by Anopheles culicifacies and Anopheles fluviatilis. This study will help in assigning chloroquine-resistant P. falciparum sporozoite transmission potential of malaria vectors and suggest that by adopting the mentioned methodologies, we can detect the spreading of the drug-resistant P. falciparum in its transmission. This approach of studying the anophelines during regular vector collection and epidemiological analysis will give the knowledge of chloroquine-resistance pattern of P. falciparum of an area and help in devising effective malaria control strategy.

The development and evaluation of a single step multiplex PCR for simultaneous detection of Anopheles annularis group mosquitoes, human host preference and Plasmodium falciparum sporozoite presence.

The Anopheles annularis group mosquitoes, subgenus Cellia Theobald (Diptera: Culicidae), includes five recognized species: An. annularis Van der Wulp, An. nivipes Theobald, An. pallidus Theobald, An. philippinensis Ludlow and An. schueffneri Stanton. From these five, the three most common species found in Orissa were considered for this study because of their remarkable vectorial and behavioral variation and the important role they play in malaria transmission. To identify and understand their role in malaria transmission we developed a single multiplex PCR-based assay. This assay included the detection of human blood feeding habit and Plasmodium falciparum sporozoite presence. Of the 186 An. annularis mosquitoes collected, morphological character-based identification showed that 94 were An. annularis, 54 were An. philippinensis and 38 were An. pallidus. However, the multiplex PCR assay confirmed that 91 were An. annularis, 56 were An. philippinensis and 39 were An. pallidus individuals after adjustments were made for misidentified specimens in the morphological method. Anopheles annularis and An. philippinensis were found positive for human blood, and two samples of An. annularis species were positive for P. falciparum sporozoites. This one-step PCR-based method constitutes a very powerful tool in large surveys of anopheline populations.